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IFIT1 is an antiviral protein that recognises
5’-triphosphate RNA
Giulio Superti-Furga, Andreas Pichlmair, Caroline Lassnig, Christoph L
Baumann, Tilmann Bürckstümmer, Thomas R Burkard, Adrijana Stefanovic,
Keiryn Bennet, Jacques Colinge, Thomas Rülicke, et al.
To cite this version:
1 1
IFIT1 is an antiviral protein that recognises
5’-2
triphosphate RNA
3
Andreas Pichlmair
1, Caroline Lassnig
2, 3, Carol-Ann Eberle
1, Maria W Górna
1, Christoph L
4
Baumann
1, Thomas R Burkard
1, Tilmann Bürckstümmer
1, Adrijana Stefanovic
1, Sigurd Krieger
5,
5
Keiryn L Bennett
1, Thomas Rülicke
3, 4, Friedemann Weber
6, Jacques Colinge
1, Mathias Müller
2, 36
and Giulio Superti-Furga
1*7
8
1
CeMM- Research Center for Molecular Medicine of the Austrian Academy of Sciences,
9
Vienna, Austria;
2Institute of Animal Breeding and Genetics,
3Biomodels Austria and
4Institute
10
of Laboratory Animal Science, Vetmeduni Vienna, Austria;
5Department of Clinical Pathology,
11
Medical University of Vienna, Vienna, Austria;
6Institute for Virology, Philipps University
12
Marburg, Germany
13 14* Corresponding author:
15Giulio Superti-Furga
16CeMM - Research Center for Molecular Medicine of the Austrian Academy of Sciences
2 22
Abstract
23
Antiviral innate immunity relies on recognition of microbial structures. One such structure is viral RNA 24
that carries a triphosphate group on its 5'terminus (PPP-RNA). In an affinity proteomics approach with 25
PPP-RNA as bait we identified interferon induced protein with tetratricopeptide repeats 1 (IFIT1) to 26
mediate binding of a larger protein complex containing other IFIT family members. IFIT1 bound PPP-27
RNA with nanomolar affinity and required R187 in a highly charged C-terminal groove of the protein. In 28
the absence of IFIT1 growth and pathogenicity of PPP-RNA viruses were severely increased. In contrast, 29
IFITs were dispensable for clearance of pathogens not generating PPP-RNA. Based on this specificity and 30
the high abundance of IFITs after infection we propose that the IFIT complex antagonises viruses by 31
3 33
Pattern recognition receptors (PRRs) sense molecular signatures associated with microbes1. Viral nucleic
34
acid delivered and generated during the viral life cycle can activate PRRs to initiate the innate antiviral 35
defence 2. Recently, triphosphorylated RNA (PPP-RNA), which is constituent of genomic, antigenomic
36
and certain transcript RNAs associated with some viruses like influenza and vesicular stomatitis virus, 37
was identified as one such component that can be recognised by the innate immune system 3-5. Binding of
38
PPP-RNA to the PRR Retinoic acid inducible gene-I (RIG-I) mediates activation of a signalling cascade 39
that culminates in the expression of type-I interferon (IFN-α/β) and other cytokines 5, 6. Most likely
40
through evolutionary pressure exerted by the innate immune system, some viruses evolved sophisticated 41
mechanisms to avoid presentation of PPP-RNA7, 8. These viruses are often sensed through atypical 42
nucleic acids components such as long double-stranded RNA (dsRNA), which activates Melanoma 43
differentiated associated gene-5 (Mda5) to initiate expression of IFN-α/β 5, 9. Beside their
interferon-44
inducing capabilities, viral RNAs are known to trigger additional cellular functions that are unrelated to 45
transcriptional control of cytokine expression 2. Thus, the cellular machinery not only discriminates
46
between host and invading molecules but often selectively targets the same structures as part of an 47
antiviral program execution. Several interferon-stimulated proteins only reveal their antiviral potential 48
after binding to dsRNA 10. However, some viruses like influenza and Rift valley fever virus appear to 49
generate only limited amounts of long dsRNA 3, 11, yet they are antagonised by IFN-α/β consistent with
50
the notion that alternative viral nucleic acid structures like PPP-RNA may be key to inhibiting their 51
replication. Moreover, there are many early and strongly IFN-α/β-induced proteins that have unclear 52
molecular function and could in principle participate in the machinery involved in engagement of viral 53
nucleic acid some of which have been revealed through viral or host genetics 12-14. In particular, little is
54
known about the cellular repertoire of proteins that have the potential to bind the type of PPP-RNA that is 55
generated during viral infection. Here we used an unbiased proteomic-centred survey to identify cellular 56
proteins that engage microbial structures 15 and report the identification and functional characterisation of
4 a class of proteins binding to PPP-RNA.
5 59
Results
60
IFIT1 and IFIT-5 are PPP-RNA binding molecules 61
We used agarose beads coupled to PPP-RNA (mimicking viral RNA) or the same RNA not containing a 62
triphosphate group (OH-RNA), which is known not to activate the innate immune system 3, to affinity
63
purify potentially interacting proteins from HEK293 cells that were or were not pre-treated with 64
recombinant Interferon β (IFN-β). Pulled-down proteins were identified by mass spectrometry 65
(Supplementary Fig. 1a). The proteins predominantly precipitated with PPP-RNA from interferon 66
treated cells were interferon stimulated proteins with tetratricopeptide repeats (IFIT) 1-5 (Supplementary 67
Fig. 1b). A double-logarithmic plot of the spectral counts as well as the exponential modified protein 68
abundance index (emPAI) 16 confirmed the specific IFN-β dependent enrichment of IFIT1, IFIT2 and
69
IFIT3 (Fig. 1a, Supplementary Fig. 1c). IFITs were expressed at low levels at steady-state but highly 70
induced by type-I interferon (IFN-α/β) and virus infection 17. 16 h after treatment with 1000 U/ml
hIFN-71
β, HeLa cells contained 216 pg/µg IFIT1 roughly corresponding to 2.4 million copies per cell (Fig. 1b), 72
while in 293T cells the IFIT1 levels were 126 pg/µg, corresponding to some 1.4 million copies 73
(Supplementary Fig. 2), placing IFIT1 amongst the most abundant cellular proteins 18. The IFIT protein
74
family contains four known human (IFIT1, IFIT2, IFIT3, IFIT5) and three mouse members (Ifit1, Ifit-2, 75
Ifit-3) (Supplementary Fig. 3). IFITs consist mainly of tetratricopeptide repeats (TPRs) but no annotated 76
nucleic acid binding domain 17. We tested the binding of IFITs to RNA by using PPP-RNA-coated beads
77
to precipitate human IFITs from IFIT overexpressing 293T cells or using recombinant protein expressed 78
in bacteria. Overexpressed and recombinant IFIT1 and IFIT5, but little IFIT2 and no IFIT3, associated 79
with PPP-RNA beads (Fig. 1c, d). The two members of the family that best bound to PPP-RNA, IFIT1 80
6
hypothesised that IFIT2 and IFIT 3 associate with PPP-RNA indirectly and be part of a molecular 82
complex that only assembles after IFN-α/β induction. 83
IFITs form an interferon-dependent multiprotein complex 84
To study the putative cellular complex assembling around the IFIT family members we performed affinity 85
purification-mass spectrometry (AP-MS) analysis using IFIT1, IFIT 2 and IFIT 3 as baits. We expressed 86
IFIT1, IFIT 2 and IFIT 3 in doxycycline-inducible HEK-FlpIN cells in the presence or absence of IFN-87
α/β. Doxycycline treatment of HEK-FlpIN cells elicited expression of IFIT1 protein that was comparable 88
to the endogenous levels measured in cells treated with 50 to 500 U/ml of hIFN-β (Supplementary Fig. 89
4a). Moreover, using a green fluorescent protein (GFP)-expressing isogenic cell line it was possible to 90
ascertain that expression in this system is highly homogenous among the cell population (Supplementary 91
Fig. 4b). Protein complexes were purified by tandem affinity purification and analysed by Liquid 92
Chromatography-Mass Spectrometry (LC-MSMS )19, 20. IFIT proteins interacted with a limited number of
93
cellular proteins in unstimulated cells (Supplementary Fig. 5; IntAct database 21 identifier IM-15277).
94
However, IFN-α/β treatment drastically changed the interaction profile in terms of number of identified 95
proteins and peptide count per protein. In purifications from IFN-α/β stimulated cells, IFIT2 and IFIT3 96
co-purified with IFIT1 with high enough sequence coverage to suggest a stochiometric interaction among 97
the three proteins (Table 1). IFIT5 did not co-precipitate with any other IFIT protein. IFITs do not require 98
IFN-α/β-induced factors to bind to each other since tagged versions of IFIT proteins co-precipitated after 99
overexpression of single proteins (Supplementary Fig. 6a). Similarly, recombinant purified IFIT1 and 100
IFIT2, IFIT1 and IFIT3 but not IFIT1 and IFIT5 associated in gel filtration experiments, suggesting a 101
direct interaction at a roughly 1:1 ratio (Fig. 2a, Supplementary Fig. 6b), consistent with the results 102
obtained by mass spectrometry on cellular complexes. Compiling the individual interaction profiles into a 103
network analysis revealed several interesting features. First, IFN-α/β induced a dramatic change in the 104
7
only after IFN-α/β induction when they find partners. Also the topology of the network is affected by 106
IFN-α/β stimulation with the dramatic increase of proteins interacting with all three baits from 1 node to 107
14 nodes (Fig. 2b, red dots). At the same time the high degree of connectivity after IFN-α/β validated the 108
quality of the analysis, as contaminants would interact also in non-induced cells. Importantly, the network 109
also suggested that a few inducible components, in this case mainly the IFIT members, may exert their 110
function by recruiting cellular proteins to assemble IFN-triggered cellular machines (Fig. 2c). 111
Interestingly, IFIT1B, a poorly characterised member of the IFIT family interacts with both IFIT1 and 112
IFIT3 making it a possible component of the larger complex or of a subcomplex worth investigating in 113
the future (Fig. 2c). Among the group of proteins interacting with more than one IFIT member are hnRNP 114
components, known to bind RNA and regulate transport and translation, small nuclear Ribonucleoprotein 115
particle (SNRP) components, RNA binding proteins involved in RNA processing, as well as polyA-116
binding proteins. While we cannot exclude that these proteins co-precipitate through binding an RNA 117
species that simultaneously binds to IFITs, this is unlikely as it would have to be via an IFN- α/β-118
inducible RNA. Overall the protein complex suggests a role of IFIT family members in RNA biology. In 119
future, it may be worth investigating the contribution of several members of the IFIT interactome in the 120
antiviral program. Here we initially focus on IFIT1 being the component mediating association of the 121
IFIT complex to PPP-RNA. 122
Molecular basis for IFIT1 interaction to PPP-RNA 123
Interferon-stimulated proteins partially re-distribute upon engagement of the respective viral ligands 22.
124
We examined the subcellular localisation of murine Ifit1 in IFN-β stimulated NIH3T3 cells after 125
transfection of biotinylated PPP-RNA or OH-RNA. Ifit1 is equally distributed in IFN-β treated cells and 126
re-localises to discrete intracellular foci after stimulation with PPP-RNA in roughly half of all cells 127
examined (Fig. 3a). In contrast, only a small fraction of cells showed relocalisation of Ifit1 after 128
8
To further assess the association of PPP-RNA with IFIT1 we investigated the requirement for 130
triphosphates in RNA precipitations comparing cells expressing c-Myc-tagged IFIT1 to cells expressing 131
GFP-RIG-I as positive control. In both cases PPP-RNA was considerably more efficient than its OH 132
counterpart in purifying the two proteins (Fig. 3b). Similarly, PPP-RNA efficiently and specifically 133
purified endogenous IFIT1 from both interferon treated HEK293 cells and mouse embryonic fibroblasts 134
(MEFs) (Fig. 3c), suggesting that the PPP-RNA binding property of IFIT1 is common to different cells 135
and species. To further assess the PPP-RNA binding properties of IFIT1 we took advantage of 136
Escherichia coli purified proteins in gel mobility assays. IFIT1 but not IFIT3 caused mobility retardation
137
of a PPP-RNA and not a OH-RNA probe (Fig. 3d). Antibodies directed against the recombinant IFIT1 138
caused an increased retardation in mobility confirming that IFIT1 is a major component of the retarded 139
complex. IFIT1 contains no recognised RNA binding domain and to identify a potential interaction 140
mechanism we relied on homology modelling with the closest homologue in the PDB database, O-linked 141
β-N-acetylglucosamine transferase (PDB code 1w3b; Fig. 3e, Supplementary Fig. 7) 23. The model
142
shows a superhelical structure of the several tetratricopeptide repeats with an extended groove winding 143
along the longitudinal axis of the protein (Fig. 3e, Supplementary Fig. 7). Large patches of positively 144
charged surfaces (blue) can be seen both in the central part of the groove and in C-terminal part of the 145
protein. We identified individual residues different between IFIT1 and IFIT3, mutated these residues into 146
the IFIT3 identity and tested for PPP-RNA binding. Only R187H showed a significant loss of association 147
(Fig. 3f, Supplementary Fig. 8a). In these experiments tagged IFIT3 was co-expressed, allowing the 148
demonstration that without a functional PPP-RNA binding moiety, as in the case of the IFIT1(R187H), 149
IFIT3 will not co-purify with PPP-RNA (Fig. 3f). Importantly, IFIT1(R187H) maintained its ability to 150
associate with IFIT3 as shown by co-immunoprecipitation experiments and gel filtration (Supplementary 151
Fig. 8b, c) indicating that the R187H mutation is not associated with a major folding problem of the 152
protein. To quantify the binding capabilities of wild-type (wt) IFIT1 compared to the mutant we used 153
PPP-RNA- and OH-RNA-coated ELISA plates and found that only the intact IFIT1 displayed a 154
9
affinities we then used surface plasmon resonance and measured an estimated binding constant of 156
recombinant IFIT1 for PPP-RNA of 242 nM and a 10-20 fold lower affinity of IFIT1 for OH-RNA (3.14 157
mM) or IFIT1(R187H) for PPP-RNA (4.36 mM) and OH-RNA (2.64 mM) (Fig. 3h). Altogether these 158
experiments demonstrate that IFIT1 has the ability to bind directly and specifically to PPP-RNA. 159
Moreover, the data strongly suggest that TPR motifs, such as the ones present in the IFIT1 protein, have 160
the ability to convey specific interactions with nucleic acids, further expanding their well characterised 161
protein-protein interaction property 24.
162
Sequestration of PPP-RNA by IFIT proteins 163
Previous studies suggested that IFIT1 suppresses in vitro translation through binding eIF3e 17, 25. While
164
we were able to confirm an overall negative effect of IFITs in PPP-RNA programmed translation assays 165
using rabbit reticulocyte lysates 17, 25, in our experiments it strongly correlated with the RNA-binding
166
properties of the different IFITs. Since the commonly used templates generated by in vitro transcription 167
are not capped and contain a triphosphate group at the 5’ end our findings suggest a simple mechanism 168
involving PPP-RNA sequestration for the observed inhibitory effects. Accordingly, IFIT1 and IFIT5, the 169
only two family members capable of binding PPP-RNA directly, most efficiently interfered with the assay 170
(Fig. 4a). If sequestration was indeed involved it should be antagonised by excess template. To directly 171
test this hypothesis we increased the amount of template RNA and assayed the ensuing translation 172
efficiency. The inhibitory effect of IFIT1 was inversely proportional to the amount of template RNA used 173
in these assays (Fig. 4b) and depended on the presence of triphosphates on the 5’ end (Fig. 4c). To finally 174
prove that it is PPP-RNA binding that lies at the center of the inhibitory effect we used the IFIT1 mutated 175
in R187 to find that IFIT1(R187H) was indeed less effective (Fig. 4d). To further exclude any possible 176
interference with the translational machinery based on protein-protein interaction properties we choose 177
the translational assay obtained from wheat germ extract. IFIT1 had an inhibitory effect comparable to the 178
one observed with rodent-derived extracts (Fig. 4e). As evolutionary distance between plants and animals 179
10
mechanistically meaningful effects on the translational machinery through a protein-protein interaction 181
extremely unlikely. Altogether, this set of data is compatible with the ability of IFIT1 to sequester PPP-182
RNA and offers a simple mechanism for the negative effects in translational assays. 183
To test whether IFIT1 has the ability to engage viral RNA also in infected cells, we precipitated tagged 184
IFIT1 or tagged GFP as control from cells infected with vesicular stomatitis virus (VSV) or influenza A 185
virus (FluAV) and tested the association of viral RNA. IFIT1 but not GFP precipitated viral RNA (Fig. 186
4f, g) suggesting that IFIT1 can also bind and potentially sequester viral RNA in cells. 187
Antiviral effects of IFIT family members 188
As IFIT1 participates in a protein complex containing stoichiometric amounts of IFIT2 and IFIT3, to test 189
antiviral activity we addressed all three family members and also where appropriate the IFIT1 ortholog 190
IFIT5. Consistent with the requirement for the formation of a protein complex, overexpression of 191
individual family members did not impair virus growth (Supplementary Fig. 9 and data not shown). 192
siRNA knockdown of IFIT members in HeLa cells effectively and specifically caused reduction of 193
transcript levels and expression of the cognate protein (Fig. 5a, b, Supplementary Fig. 10 a-d) but did 194
not influence induction of IFN-β mRNA (Supplementary Fig. 10e). Loss of IFIT family members led to 195
an increase in growth of VSV, VSV-M2 (mutated in the Matrix protein, M51R leading to IFN-β 196
induction) and Rift valley fever virus (RVFV Clone13) to different degrees, with IFIT1 and IFIT2 being 197
most efficient (Fig. 5c-e, Supplementary Fig. 11a-e). In contrast, growth of encephalomyocarditis virus 198
(EMCV) was not significantly affected by the siRNA treatments (Fig. 5f, Supplementary Fig. 11f), 199
consistent with the notion that EMCV does not generate PPP-RNA during its replication cycle 28. Similar
200
to other PPP-RNA generating viruses, also the replication of FluAV, as measured by activation of a 201
polymerase-I promoter read-out, increased in the absence of IFIT1, IFIT2 and IFIT3, suggesting that the 202
entire IFIT1 complex is involved in antiviral activities against influenza (Fig. 5g). Collectively our data 203
11
producing viruses. The contribution of the different family members may differ depending on the nature 205
of the microbial agent. As the affinity of IFIT1 to PPP-RNA constitutes a central feature of the IFIT1 206
complex, we directly tested its importance for antiviral activity. For this we expressed siRNA-resistant 207
versions of wt IFIT1 and the PPP-RNA binding mutant IFIT1(R187H) (Fig. 5h), respectively, in cells 208
that were treated with siRNA against IFIT1. We used as read-out the FluAV polymerase-I dependent 209
transcriptional assay to observe that the PPP-RNA binding impaired IFIT1(R187H) mutant was 210
considerably less able to constrain viral replication as compared to wt IFIT1 (Fig. 5i). Taken together 211
these data clearly show that the requirements for an efficient antiviral activity include the presence of all 212
three family members, IFIT1, IFIT2 and IFIT3, and the PPP-RNA binding capability of IFIT1. 213
Ifit1 displays antiviral activity in vivo 214
Mice should be a particularly suitable model system to study IFIT activity since mouse Ifit1 is the only 215
family member binding PPP-RNA and knockdown cell lines using shRNA against Ifit1 were impaired in 216
their ability to contain virus growth in the presence of IFN-β (Supplementary Fig. 11 g-j). We generated 217
mice with a deletion in the Ifit1 gene (Fig. 6a). Ifit1 deficiency was confirmed by quantitative PCR (Fig. 218
6b) and immunoblotting of lysates from IFN-β stimulated MEFs (Fig. 6c). The absence of mouse Ifit1 219
was not due to defective signalling downstream of the type-I interferon receptor since the interferon 220
responsive protein DAI 29 was induced upon IFN-β treatment (Fig. 6c). Under specific pathogen-free
221
(SPF) conditions, mice lacking Ifit1 showed no phenotypic abnormalities and were undistinguishable 222
from wt C57BL/6 mice. 223
IFITs have been proposed to regulate cytokine expression 30, 31. However, Ifit1 deficiency did not change
224
phosphorylation of the transcription factor IRF3 in response to transfection of innate immune stimuli 225
(Supplementary Fig. 12a). Transfecting PPP-RNA, viral RNA derived from VSV particles (vRNA), 226
poly-I:C, interferon stimulatory DNA (ISD) or poly-dA:dT, or activation of the TLR pathway through 227
12
vivo bone-marrow, bone marrow-derived macrophages and bone marrow-derived dendritic cells (Fig. 6d,
229
Supplementary Fig. 12b-g). Neither was the induction of IFN-α/β and IL-6 protein by viral infection 230
affected by Ifit1deficiency (data not shown). We therefore concluded that Ifit1 is dispensable for 231
induction of antiviral cytokines. In contrast, Ifit1-deficient cells allowed consistent higher VSV 232
accumulation compared to wt counterparts at three different time points tested (Fig. 6e). EMCV infected 233
MEFs showed equal viral loads irrespective of the genetic status of the Ifit1 gene (Fig. 6f). 234
To establish an antiviral function of Ifit1 in vivo we infected Ifit1 knockout mice with VSV. At all doses 235
tested, Ifit1 deficient mice showed significantly reduced survival as compared to control mice (Fig. 6g, 236
Supplementary Fig. 13a and data not shown), suggesting that control of VSV infection required Ifit1 237
also in vivo. In contrast, absence of Ifit1 did not affect viability of mice infected with EMCV (Fig. 6h, 238
Supplementary Fig. 13b). Similar to EMCV, Ifit1 seemed to be dispensable for the clearance of Listeria 239
monocytogenes, a bacterium known to predominantly engage DNA-sensing pathways 32, 33 (Fig. 6i,
240
Supplementary Fig. 13c). We concluded that in vivo, Ifit1 manifests a critical activity against VSV and 241
presumably other PPP-RNA-expressing viruses but not against the other pathogens tested here. 242
Overall we conclude that the IFIT proteins contribute to an executing branch of the PPP-RNA innate 243
immunity molecular network. While RIG-I represents the PPP-RNA sensing module that signals towards 244
type-I interferon production, interferon causes a feed-back mechanism that ensures the arming of cells 245
with PPP-RNA-binding antiviral proteins, such as IFIT1, IFIT5 and the protein complexes that they form. 246
(Supplementary Fig. 14). 247
13 249
Discussion
250
IFIT1 demands the regular attention of immunologists, since it is encoded by one of the most abundantly 251
IFN-α/β induced mRNAs. So far most evidence has been gathered for it being a general inhibitor of 252
protein translation 17. Recently, however, elegant studies using viruses defective in their ability to
253
methylate mRNA CAP structures at the 2’O-position and Ifit1 and Ifit2 deficient mice identified an 254
intriguing correlation between specific 5’nucleic acid conformations and Ifit function 14 for which the
255
present study offers a mechanistic rationale. While IFIT1 is shown here to bind PPP-RNA, IFIT2 and 256
IFIT3 also have a virus-containing function and all three proteins form a complex that contains yet other 257
family members as well as other RNA-binding proteins. This raises the possibility that the IFIT complex 258
represents multiple RNA-binding valencies able to recognise and counteract a yet to be determined 259
spectrum of microbes. The IFIT versatility may well reside in the modular use of TPRs, shown here to 260
have nucleic acid binding capability, in analogy to the role of leucine-rich repeats that confer binding 261
plasticity to another family of PRR, namely the Toll-like receptors. Unlike these, IFITs are strongly 262
induced during infection and reach expression levels beyond a million copies per cell. This abundance, 263
rather than with the signalling roles of receptors, may be more compatible with an executing function. We 264
therefore suggest a general model whereby IFIT proteins exert their antiviral activity by physically 265
engaging microbial elements. In particular the present work focuses on the 5’conformation of RNAs such 266
as it is present on the genomic, antigenomic and some transcripts of certain virus species. While members 267
of the RIG-I helicases represent the PPP-RNA binding components of the sensing and interferon 268
induction branch of the innate immunity molecular network, we here propose that IFIT family members 269
represent the PPP-RNA binding component of an executing antiviral branch of the network. The final fate 270
of the PPP-RNA physically sequestered by the IFIT complex remains to be elucidated. Sequestration of 271
viral components has been described before in the case of orthomyxovirus resistance (Mx) proteins 272
known to physically inhibit assembly of viral particles though binding viral proteins 34. Some viruses
14
generate large amounts of small triphosphorylated leader-RNAs which could potentially antagonise IFIT 274
activity 35. We suggest that similarly to the diverse set of proteins sensing the variety of PAMPs and 275
triggering the anti-pathogen response, also the abundant proteins executing the response itself need to 276
maintain specificity for defined pathogen structures to limit interference with vital host processes. 277
15
Database accession numbers
279
Mass spectrometry data presented in Figure 2 was deposited in the IntAct database 21, identifier:
IM-280
16 282
Table 1
283
Bait protein
IFIT1 IFIT2 IFIT3 No IFN + IFN No IFN + IFN No IFN + IFN
IFIT1 19 34 5 29 14 32
IFIT2 0 17 24 25 0 19
IFIT3 0 29 5 25 32 28
IFIT5 0 0 0 0 0 0
284
HEK-FlpIN cells were stimulated with 1 µg/ml doxycycline for 24 h to induce expression of IFIT1. Cells 285
were left untreated or treated overnight with approximately 1000 U/ml IFN-α/β that was generated by 286
transfecting HEK293 cells with poly-I:C. Protein complexes isolated by tandem affinity purification were 287
analysed by LC-MSMS. The table shows number of identified IFIT peptides in precipitations of IFITs 288
17 290
Figure Legends
291
Figure 1: Identification of an IFN-α/β-induced IFIT containing complex as a PPP-RNA binding 292
entity 293
(a) HEK293 cells were left untreated or treated with 1000 U/ml IFN-β overnight. Cells were lysed and 294
incubated with PPP-RNA or OH-RNA coupled to streptavidin beads. After precipitation, bead-associated 295
proteins were eluted, separated by 1D SDS PAGE electrophoresis and whole lanes analysed by Liquid 296
Chromatography-Mass Spectrometry (LC-MSMS). Identified proteins are represented as dots with 297
detection strength (log of spectral count) in OH-RNA pull downs (x-axis) and PPP-RNA pull downs (y-298
axis), both in IFN-β stimulated conditions. Red dots represent proteins with no detection in the absence of 299
IFN-β in both OH-RNA and PPP-RNA pull downs. IFIT proteins are by far the strongest hits. IFIT5 is 300
gray due to detection in the pull down done in the absence of IFN-β priming. Data from four experiments 301
is shown. (b) 106 HeLa cells were treated with the indicated amount of recombinant IFN-β for 16h and
302
the lysates, alongside a recombinant IFIT1 standard, were analysed by immunoblotting for IFIT1 and 303
tubulin. The signal was quantified using infrared imaging. The cellular copy number of IFIT1 in per HeLa 304
cells treated with 1000 U/ml IFN-β was determined to be 2,4 * 106. One of two experiments done in
305
duplicate is shown. (c, d) Lysates from 293T cells transfected with plasmids for c-Myc-tagged IFITs (c) 306
and E. coli expressing His-GST-tagged IFITs (d) were used for affinity precipitation with PPP-RNA and 307
associated proteins analysed by immunoblotting. 308
309
18
(a) Recombinant IFIT proteins and their binary complexes were analyzed by size-exclusion 311
chromatography. Shown are overlaid elution profiles from Superdex 200 10/300 GL column (the void 312
volume is ~8.3 ml), and the indicated peak fractions were analyzed by SDS-PAGE followed by 313
coomassie staining. His-tagged IFIT1 binds His-GST-tagged IFIT2. (b, c) Network analysis of the IFIT 314
protein complex based on data described in Table 1. (b) The IFIT proteins (large balls) in absence of IFN-315
α/β stimulation (left) are interacting with fewer proteins (small balls) whereas upon IFN-α/β stimulation 316
IFITs recruit many new partners. Interactions between IFIT-1, -2, and -3 are also stronger. Proteins 317
identified by all IFITs are shown in red. (c) Protein interaction network for the IFN-α/β stimulated 318
condition and annotated protein functions using Gene Ontology (GO) molecular functions and manual 319
curation. Obvious non-specific proteins or contaminants were removed (keratin, albumin from MS BSA 320
quality control runs, and MCC12 and PCCAB which bind to the Strep-tactin affinity resin in high 321
abundance). Many of the shared IFIT partners have the ability to bind to RNA (red) and some are 322
involved in mRNA translation (green). IFIT bait proteins are shown in blue. 323
324
Figure 3: Triphosphate-dependent RNA-binding of IFIT1 requires an Arginine at position 187 325
(a) Ifit1 redistribution (white arrows) in IFN-β - treated NIH 3T3 cells transfected with biotinylated PPP-326
RNA and OH-RNA for 3 h. Shown is the average % relocalisation of Ifit1 (+/- standard deviation) in 100 327
randomly selected cells in two independent experiments. * = p < 0,05. (b, c) PPP-RNA or OH-RNA 328
beads were used for affinity purification from lysates of 293T cells expressing c-Myc-IFIT1 or GFP-RIG-329
I (b) or IFN-β treated HEK293 cells and MEFs (c). Precipitates were analysed by immunoblotting. (d) 330
Mobility shift assay of PPP-RNA and OH-RNA by recombinant His- GST-IFIT1 and -IFIT3. Where 331
indicated an antibody against GST was added. Numbers on the right indicate free probe (1), shifted probe 332
(2) and supershifted probe (3). (e) Surface charge of an IFIT1 structure model based on O-linked β-N-333
19
negative, blue is positive charge, N is N-terminus, C is C-terminus. Proteins with targeted point mutations 335
of the indicated residues were used for further functional characterisation. (f) c-Myc-tagged IFIT1 336
mutants and HA-IFIT3 were co-expressed in 293T cells and 24 h later used for affinity purification using 337
PPP-RNA as bait. (g) PPP-RNA or OH-RNA were bound to ELISA plates and incubated with the 338
indicated amounts (ng) of recombinant IFIT1 or IFIT1(R187H). RNA-asssociated proteins were detected 339
using secondary reagents. Shown is substrate conversion at OD 450, error bars show standard deviation of 340
triplicate measurements. One representative experiment of three is shown. (h) The affinity of IFIT1 and 341
IFIT1(R187H) to PPP-RNA and OH-RNA was measured by surface plasmon resonance using 342
biotinylated RNA as immobilised ligand and increasing amounts of recombinant protein. Shown are the 343
response units of the indicated combinations of binding partners with standard deviation from duplicate 344
measurements. 345
346
Figure 4: IFIT1 sequesters PPP-RNA in vitro 347
(a-d) Rabbit reticulate lysate (RRL) or (e) wheat germ extract (WGE) was supplemented with RNA 348
template expressing firefly-luciferase and recombinant IFITs or no protein was added. (a, d, e) 0.2 µg of 349
in vitro transcribed PPP-RNA template (that is commonly used in such assays) was incubated with the
350
indicated amounts of recombinant IFITs or no protein. (b) As in (a) but 0.2 µg and 0.05 µg template RNA 351
were used. (c) RNA that was not (PPP-luc) or was treated with calf intestinal phosphatase (OH-luc) was 352
supplemented together with 35 µM IFIT1, as indicated. (d) Translation of PPP-luc mRNA template in the 353
presence of 35 µM IFIT3, IFIT1 or IFIT1(R187H). (a-e) The graph shows luciferase activity after an 1 h 354
incubation period at 37 °C. Error bars show standard deviation of at least two experiments done in 355
triplicate measurements. * = p<0,05, n.s. = non significant. (f-g) HEK-FlpIN IFIT1 or HEK-FlpIN GFP 356
cells were stimulated with doxycycline for 24 h and infected with VSV-GFP and FluAV (both MOI: 5) 357
20
after precipitation (SII-IP) was analysed by qRT-PCR for VSV (f) or FluAV sequences (g). The graph 359
shows arbitrary units +/- standard deviation of duplicate measurements of one representative experiment 360
of three (f) or two (g). 361
362
Figure 5: Influence of IFIT RNA interference on virus growth 363
(a) 105 HeLa cells were transfected with 0.5 µg of the indicated IFIT expression vector and 5 nM siRNA
364
directed against the indicated IFIT family member. Expression of c-Myc-tagged proteins was evaluated 365
by immunoblot 48 h later. (b-f) HeLa cells were transfected with 5 nM siRNA for 48 h. (b) Cells were 366
stimulated with 0.25 µg PPP-RNA for 16 h and expression of IFIT1 or IFIT3 was tested by 367
immunoblotting. (c-f) siRNA treated HeLa cells were infected at a multiplicity of infection (MOI) of 0.01 368
with VSV (c), VSV-M2 (with a M51R mutation in the matrix protein) 36 (d), RVFV (Clone13) (e) or
369
EMCV (f) and virus accumulation was tested by TCID50 at 48 h (c, d, f ) and 72 h (e) after infection. 370
Graphs in (c-f) show the average of three independent experiments, error bars indicate standard deviation. 371
(g) HeLa cells were co-transfected with Pol-I ff-luc (0.1 µg), pRL-TK (0.05 µg) reporter plasmids and the 372
indicated siRNAs. 48 h later cells were left uninfected or infected with FluAV at a MOI of 1 and reporter 373
activity analysed after over-night incubation. The graph shows the ratio between firefly- and renilla 374
luciferase +/- standard deviation of one representative experiment of two done in duplicate measurements. 375
(h) HeLa cells were co-transfected with siRNA against IFIT1 or control siRNA together with plasmids 376
coding for c-Myc-tagged versions of parental or silencing-resistant IFIT1. Immunoblots 48 h after 377
transfection are shown. (i) as in (g) but plasmids coding for silencing-resistant IFIT1 were co-transfected 378
as indicated. The graph shows the ratio between firefly- and renilla luciferase +/- standard deviation of 379
one representative experiment of three done in duplicate measurements. 380
381
21
(a) Targeting strategy for mouse Ifit1. (b, c) Loss of Ifit1 in Ifit1+/+ MEFs (+/+) and Ifit1-/- MEFs (-/-) was 383
validated by PCR (b) and by immunoblotting in MEFs that were stimulated with IFN-β for 16 h (c). (d) 384
MEFs (2 * 105 cells/ml) were left unstimulated or transfected with PPP-RNA (0.4 µg/ml and 0.08 µg/ml),
385
viral RNA isolated from VSV particles (vRNA) (0.2 µg/ml), poly-I:C (1 µg/ml) or poly-dA:dT (1 µg/ml) 386
and accumulation of IFN-α/β was tested using a cell line stably containing an ISRE-luc reporter. (e, f) 387
MEFs of the indicated genotype were infected with VSV (e) or EMCV (f) at a MOI of 0.01 and virus 388
accumulation in the cell supernatant was measured by TCID50 after 48 h. Graphs show average virus 389
titers from two independent experiments. Error bars show standard deviation. * = p<0.05 tested by two 390
way Annova for two independent experiments done in hexaplicate measurements. (g-i) Survival of Ifit1 391
deficient (Ifit1-/-) (red lines) and C57BL/6 mice (Ifit1+/+) (black lines). (g) Male animals (n = 14) were
392
anesthetized with ketamine-xylazine and infected intranasally with 105 pfu of VSV and monitored twice
393
daily for survival over a two week period. Wt mice survived significantly longer than Ifit deficient 394
animals (Mantel-Cox Testp < 0.01). (h) Sex-matched Ifit1-/- and Ifit1+/+ mice (n = 17) were infected
395
intraperitoneally with 500 pfu of EMCV and monitored for survival. (i) Female Ifit1-/- and Ifit1+/+ mice (n
396
= 9) were infected intraperitoneally with 106 CFU L. monocytognes. d.p.i.: days post infection.
22 398
Acknowledgements
399
We want to thank the NIH Knock-out Mouse Project (KOMP) for providing ES cells with a targeted Ifit1 400
allele. We want to thank Lill Andersen for expansion of ES cells, Kumaran Kandasamy for bioinformatics 401
support, Michael Bergmann for providing FluAV matrix protein antibody. The work in the authors’ 402
laboratories was funded by the Austrian Academy of Sciences, the i-FIVE ERC grant to GSF, an EMBO 403
long-term fellowship to AP (ATLF 463-2008), a Marie-Curie and an EMBO fellowship to CB, DFG grant 404
We 2616/5-2 and SFB 593/B13 to FW. TR and MM are funded by the Austrian Federal Ministry for 405
Science and Research GEN-AU programme Austromouse; MM is funded by the Austrian Science Fund 406
23 408
Methods
409
Reagents, proteins and viruses 410
IFN-α and IFN-β were from PBL Interferonsource. IFN-α/β was generated by transfecting HEK293 cells 411
with poly-I:C. Expression constructs were generated by PCR amplification and cloned into pCS2-6myc-412
GW, pCDNA-HA-GW, pTO-SII-HA-GW 20 or pETG30A-GW and pETG10A-GW. Point mutations were
413
introduced by site directed mutagenesis. Pol-I ff-luc was from Georg Kochs 37. p7SK-as and pGFP-RIG-I
414
were described earlier 3. pRL-TK was from Promega. In vitro translation was done with Rabbit
415
reticulocyte lysate or Wheat germ extract (Promega) using the provided luciferase mRNA or SP6-416
polymerase transcribed luciferase mRNA as template. Strep-tacin beads were from IBA, HA-agarose 417
from Sigma, Protein G sepharose was from GE Healthcare and Streptavidin beads from Pierce. 418
Antibodies for α-tubulin and β-actin were from Alexis. Phospho-IRF3 was from Cell Signalling. IRDye - 419
conjugated anti-myc antibody, anti-mouse and anti-rabbit secondary reagents were from Rockland. 420
Streptavidin Alexa-800, Streptavidin Alexa-488 and goat anti-mouse Alexa-548 were from Molecular 421
probes. Polyclonal antibodies against rb-α−DAI, rb-α−IFIT1, ms-α−Ifit1 and rb-α−IFIT3 were generated 422
by immunisation of animals with full-length recombinant protein. RT-PCR reagents were from Qiagen. 423
Biotinylated PPP-RNA (7SK-as) was described earlier 3. PPP-RNA was dephosphorylated using Calf
424
intestinal phosphatase (New England biolabs). LPS (E.coli K12), CpG (CpG-DNA-ODN1826), poly-425
(I:C) and poly-(dA:dT) were from Invitrogen. ISD 33 was synthesised at Microsynth. vRNA was isolated
426
using Trizol (Invitrogen). For stimulation TLR agonists were added other stimuli were transfected with 427
Lipofectamine 2000 (Invitrogen) or Polyfect (Qiagen). Total IFN-α/β was measured as described 38 . IL-6
428
was measured by ELISA (BD). 429
Recombinant IFITs were expressed in E. Coli and purified on a HisTrap HP column (GE Healthcare). 430
24
AV1) 36, RVFV (Clone 13) 40 and Listeria monocytogenes (EGD) 41 were described earlier. Viruses were 432
titrated on Vero cells using the TCID50 method of Reed and Muench. 433
Cells, mice and in vivo experiments 434
293T, NIH3T3 and HEK293 cells were described earlier 3. IRF3 deficient MEFs were a gift of Thomas
435
Decker. Doxycycline regulatable HEK-FlpIN cells were from Invitrogen. MEFs were generated from 436
embryos of mated Ifit1+/- mice. BM macrophages (BMMs) were cultured in the presence of M-CSF 437
(Prepotech), BM dendritic cells (BM-DC) in presence of GM-CSF (Prepotech). Fibroblasts were kept in 438
DMEM (PAA) and primary cells cultured in RPMI (PAA) supplemented with 10 % fetal calf serum 439
(Invitrogen) and antibiotics (100 U/ml penicillin, 100 µg/ml streptomycin). For inducible transgene 440
expression HEK-FlpIN cells were treated with 1 µg/ml doxycycline for 24-48 h. For siRNA knockdown, 441
5nmol siRNA was mixed with HiPerfect (Qiagen) and added to 105 HeLa cells. 48 h later cells were used
442
for experiments. Sequences of shRNA vectors and siRNA knockdown oligos are available on request. 443
Ifit1 knockout mice were generated using ES cells clones (VGB6; C57BL/6NTac background) with a
444
targeted Ifit1 locus. ES cells were provided by the NIH-knockout mouse project (KOMP, NIH). C57BL/6 445
wild-type control mice were purchased from Charles River. All mice were kept under specific pathogen 446
free conditions according to FELASA recommendations. For EMCV infections age (9-11 weeks) and sex-447
matched mice were infected intraperitoneally, for Listeria monocytogenes (EGD) age-matched (8-11 448
weeks) females were infected intraperitonally. For VSV challenge, age-matched (8-11 weeks) male mice 449
were anesthetised with ketamine-xylazine and inoculated intranasally with VSV. All animal experiments 450
were approved by the institutional ethics committee and the Austrian laws (GZ 68.205/0057-II/10b/2010). 451
RT-PCR, Immunofluorescence, gel shift assays, protein quantification 452
RNA was isolated using RNeasy kit (Qiagen) and reverse transcribed using oligo-dT primers and the 453
RevertAID RT-PCR kit (Fermentas). NIH3T3 cells were grown overnight on coverslips and stimulated as 454
25
Alexa-548, Alexa-488-Streptavidin and DAPI. Images were acquired with a Leica AF6000 deconvolution 456
microscope. For gel shift assays 200 ng biotinylated 7SK-as RNA 3 supplemented with Alexa-800-457
Streptavidin was incubated with 12,5 µg recombinant His-GST-IFIT1 or His-GST-IFIT3 protein solved in 458
PBS supplemented with RNAsin (Promega) (1:20), DTT (final volume 400 mM) and 100 mM NaCl. 459
Where shown, GST antibody (1 µg) was added. Samples were run on a 1 % Agarose gel and RNA was 460
visualised using a LI-COR Odyssee system. To estimate the protein copy number of IFIT1 in cells, 461
recombinant IFIT1 was used as calibration standard and compared to lysates of IFN-β stimulated HeLa 462
and 293T cells. The signal intensity on western blots was quantified using a LI-COR Odyssee system. 463
Affinity purifications and measurements, mass spectrometry and homology modelling 464
For RNA precipitation 5 µg PPP-RNA or OH-RNA (both 7SK-as) were added to streptavidin resin, and 465
incubated with 6 mg of HEK293 cell lysate for 60 minutes. Beads were washed three times in TAP-buffer 466
(50 mM Tris pH 7.5, 100 mM NaCl, 5 % (v/v) glycerol, 0.2 % (v/v) Nonidet-P40, 1.5 mM MgCl2 and
467
protease inhibitor cocktail (Complete, Roche)), proteins eluted by boiling in SDS sample buffer and 468
analysed by one-dimensional SDS-PAGE. Entire gel lanes were anaylsed by mass spectrometry using a 469
hybrid LTQ-Orbitrap XL (ThermoFisher Scientific) or a quadrupole time-of-flight mass spectrometer 470
(QTOF Premier; Waters) coupled to an 1100/1200 series HPLC (Agilent Technologies) with an analytical 471
column packed with C18 material. Data generated by LC-MSMS was searched against 472
UniProtKB/SwissProt version 57.12 42 integrating Mascot 43 and Phenyx 44 search engines. A false
473
discovery rate of less than 1 % on the protein groups was estimated. HEK-FlpIN cells and isolation of 474
protein complexes for LC-MSMS analysis is described elsewhere 20. 293T cells were transfected with
475
respective expression plasmids for 48 h and lysates used for immunoprecipitation using HA-agarose or 476
RNA-coated beads. For surface plasmon resonance measurements biotinylated 7SK-as RNA was loaded 477
on a streptavidin coated SA sensor chip (GE Healthcare) and probed with recombinant wild-type or 478
IFIT1(R187H) diluted in running buffer (0.01 M Hepes, pH 7.4, 0.25 M NaCl, 0.005 % surfactant P20). 479
26
integration functions of the BIAevaluation 3.1 software package. To determine the dissociation constant 481
(KD) the equilibrium-state binding values were plotted as a function of the applied protein concentrations 482
and fitted to first-order kinetics assuming a monovalent RNA-protein interaction. Comparative modelling 483
was done using the I-TASSER server (http://zhanglab.ccmb.med.umich.edu/I-TASSER/) 45 to obtain a
484
model for full-length IFIT1. The model was based on the structure of O-linked β-N-acetylglucosamine 485
transferase (PDB code 1w3b), with 17 % sequence identity. Surface charge potential was calculated by 486
27 488
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Figure 1
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PPP-RNA Precipitation (Log SC)
● ● ● ● ● ● ● ● ● ● ● ●● ●●● ● ● ●●● ● ●● ●● ●● ●●● ● ● ● ●●●● ● ●●●● ● ● ● ● ●● ●●● ● ●●●●●● ● ● ● ● ●●● ● ● ●● ● ● ● ●●●●●● ● ● ●●●● ● ● ●● ●● ● ●● ●●●● ● ● ● ●●●● IFIT1 IFIT2IFIT3 IFIT5 b c IB: c-Myc IFIT1 IFIT2 IFIT3 IFIT5
Input IP: RNA 293T
HeLa + IFN-β (U/ml)
1000333 111 37 12,3 0 IB: tubulin IB: IFIT1 Recombinant IFIT1 (ng) 30 20 10 5 2,5 1 0,5 0,25 independent dependent ● ●IFN-β Input IP: RNA IB: HIS E. coli d
Figure 3
b d e IB: c-Myc IB: GFP c-Myc-IFIT1 GFP-RIG-I PPP-RNA IP Input OH-RNA 293T PPP-RNA OH-RNAIFIT3IFIT1IFIT1 + IFIT3 AB no protein no proteinIFIT1
1 2 3 N R187 K429 o 155 R315K317 K348 R386 C + _ f
Input IB: c-Myc
IB: HA IP: PPP-RNA IB: c-Myc IB: HA HA-IFIT3 + + + + + + c-Myc IFIT1 R187H Wt R315S/K317MK348ER386NK429L h OH-RNA Ifit1 PPP-RNA Ifit1 % relocalisation 0 20 40 60 * PPP-RNA OH-RNA a
IB: human IFIT1
IFNβ _ + + +
Figure 4
0 2 4 6 8 IFIT1 IFIT3 IFIT1 (R187H) * 0 2 4 6 8 10Luciferase activity (10 RLU)
IFIT1 IFIT2 IFIT3 IFIT5 0 70 µM 35 µM 0 2 4 6 8 * OH-luc IFIT1_ + _ + + _ + _ PPP-luc + + _ _ b d f g SII-IP Input -12 -11 -10 -09 -08 -07 IFIT1GFP 10-06 FluAV (AU) SII-IP Input -12 -11 -10 10-09 IFIT1 GFP VSV (AU) 10 10 10 10 10 10 10 10 10 a RRL RRL RRL e IFIT1 (µM)0 35 9 0 3 6 9 12 WGE 0.0 0.5 1.0 1.5 Luciferase activity (fold) 0,2 µg RNA 0,05 µg RNA 35 µM17,5 µMNo IFIT1 n.s. * * RRL c 4
Luciferase activity (10 RLU)
3
Luciferase activity (10 RLU)
4
Luciferase activity (10 RLU)
Figure 5
a b c si IFIT1 si IFIT2 si IFIT3 si IFIT5 si CtrlIFIT1 IFIT2 IFIT3 IFIT5
c-Myc-IB: c-Myc si IFIT1 IB: IFIT1 si Ctrl IB: tubulin + + _ _ IB: IFIT3 IB: tubulin si IFIT3 si Ctrl + + _ _
si Ctrlsi IFIsi T1IFIsi T2IFIT3si IFIT5
No si RNA 104 105 106 107 108 VSV (TCID /ml) si Ctrlsi IFIT1 si IFI T2 si IFI T3 si IFI T5 No si RNA 104 105 106 107 10f 8
si Ctrlsi IFIsi T1IFIT2si IFIT3si IFIT5
No si RNA 104 105 106 107 108 104 105 106 107
si Ctrlsi IFIsi T1IFIT2si IFIT3si IFIT5
No si RNA d e g si IFIT1 Pol-I ff-luc/ren-luc IB: IFIT1 0 2 4 6 8 10 IFIT1 (Wt) IFIT1 (R187H) IB: c-Myc + _ _+_+_+ + _ _ +_ _ ++ si Ctrl FluAV i 0.0 0.5 1.0 1.5 2.0 Pol-I ff-luc/ren-luc
si Ctrlsi IFIT1si IFIT2si IFIT3
Figure 6
a e Ifit1 -/-mIFN-β- + - + IB: Ifit1 IB: DAI IB: β-actin +/+ 1003 1004 1005 1006 1007 1008 1009 24h 48h 72h Time (h) 1004 1005 1006 1007 1008 1009 1010 VSV ( TC ID /m l) 24h 48h 72h * * * 0 2 4 6 8 10 12 14 0 20 40 60 80 100 120 Ifit1 Ifit1Time after infection (d)
su rv iv al (%) VSV 0 20 40 60 80 100 120 EMCV 0 2 4 6 8 10 12 14 0 20 40 60 80 100 120 L. monocytogenes 0 2 4 6 8 10 12 14 g h i
lac Z -polyA PGK-neo Ifit1 b 50 150 Ifit1 Ifit3 _ _ +/+ +/+_ _ -10 -08 -06 AU (log 10) c 0 5 10 15 Mock PPP-RNA vRNA poly-(I:C) poly-(dA:dT) IFN-α/β (10 U/ml) d f / / Ifit1+/+ Ifit1_ _/ Ifit1 +/+ Ifit1_ _/ Ifit1 +/+ Ifit1_ _/ 3 Time (h) +/+ _ _/ Ifit1 Ifit1+/+_ _/ Ifit1 Ifit1+/+_ _/
Time after infection (d) Time after infection (d)
